Our main vaccine platform is based on recombinant vesicular stomatitis virus (rVSVs), a live-attenuate vaccine approach. Over the years we have generated several rVSVs expressing the glycoproteins (GP) of representative isolates of all ebolavirus species: Sudan ebolavirus, Zaire ebolavirus, Ta Forest ebolavirus, Bundibugyo ebolavirus and Reston ebolavirus. Additionally, we generated rVSVs expressing the GPs of two isolates of Marburg virus (MARV), Musoke and Angola. All vaccine vectors have been extensively characterized in cell culture and their protective efficacy has been evaluated at least in animal models (rodents, nonhuman primates) largely against homologous challenges. In response to the West African Ebola epidemic, rVSV-EBOV vaccine candidate was fast-tracked and shown to be safe and immunogenic in humans. Human phase II and III clinical trials with this vaccine candidate were initiated in West Africa. (summarized in Suder et al. Hum Vaccin Immunother 2018). Cross-protection among the different filovirus species is an important consideration as endemicity zones may overlap in Sub-Saharan Africa. This, however, seems difficult to achieve due to relatively high genetic variability and therefore limited cross-protective immune responses among viruses of different species and genera. In a first attempt to address this issue, we previously used a single-injection protocol with three blended vaccine vectors and demonstrated complete protection against challenge with the three homologous virus species (Geisbert et al. J Virol 2009). We have also performed another proof-of-concept study, in which we evaluated cross-protection following immunization with a single vaccine vector (rVSV-EBOV) and demonstrated partial cross-protection against challenge with a heterologous virus species (BEBOV) (Falzarano et al. J Infect Dis 2011). This demonstrates that monovalent rVSV-based vaccines may be useful against a newly emerging filovirus species; however, heterologous protection across species remains challenging and may depend on enhancing the immune responses either through booster immunizations or through the inclusion of multiple immunogens. Overall, we can conclude that single monovalent rVSV vaccine vectors can provide partial cross-protection in cases of challenge virus species that are genetically more closely related. As mentioned above, one approach to overcome this limitation is the use of blended monovalent rVSV vaccine vectors, which provide broader protection against homologous and partial protection against certain heterologous challenges. This approach can be immediately implemented if needed. Another approach to overcome the limitations in cross-protection is the use of multivalent rVSV vaccine vectors. In a proof-of-concept study in hamsters protection against ZEBOV and Andes virus (ANDV) challenge was demonstrated using a single rVSV vector expressing both the ZEBOV GP and the ANDV glycoprotein (Tsuda et al. J Infect Dis 2011). This data showed that the use of bivalent rVSV vectors is a feasible approach to vaccination against multiple pathogens. EBOV GP provides targeting to important immune cells such as monocytes/macrophages and dendritic cells. Using this favorable targeting, we have generated further bivalent rVSV vaccines to proof the concept of immune enhancement through EBOV GP (Prescott et al., Vaccine 2015). We have developed rVSV-EBOVGP vectors expressing the Nipah virus glycoproteins (G and F) (de Buysscher et al Vaccine 2014), the Zika virus preM and E proteins (Emanuel et al. Sci Rep 2018) and the influenza hemagglutinin (H5) (Furuyama et al., manuscript in preparation). In all cases we could demonstrate complete protection against homologous challenge in respective animal models. We postulate that vaccine vectors based on rVSV-EBOVGP will show enhanced protection due to favorable immune cell targeting. Following this concept, we recently have generated a RVSV-EBOVGP vector expressing the Lassa virus glycoprotein (GP); efficacy testing in animals will start soon. The generation for VSV expressing three foreign proteins (immunogens) has proven difficult as the replication capacity of such viruses is drastically reduced. However, we have successfully generated rVSV vectors expressing a transmembrane protein (replacing the VSV glycoprotein) and two soluble antigens that will be secreted during vector replication. In vitro characterization of these vectors has been completed. Efficacy testing in the appropriate rodent models has demonstrated promising results. Further studies are planned during the next fiscal year. Overall, this project has shown promise and should be continued as the rVSV approach has shown efficacy for emergency vaccination. Vectors can be generated in a short period of time (1-2 months) and a vaccine product can be available in several months (6-9 months). Thus, this platform has potential for our response capabilities to counteract emerging infectious diseases.